Advances in the knowledge of incretin hormone mechanism of action coupled with advancements in the understanding of intestinal differentiation, both at the stem cell and endocrine cell stages, have led to interest in developing sources of incretin hormone producing cells, appropriate for engraftment. One approach is the generation of functional enteroendocrine L- or K-cells from pluripotent stem cells, such as human embryonic stem cells (“hESC”) or induced pluripotent stem cells (“iPS”).
The production/secretion of glucagon-like peptide 1 (GLP-1) from intestinal L-cells or glucose-dependent insulinotropic polypeptide (GIP) from intestinal K-cells has beneficial effects for the treatment of diabetes mellitus. Incretin hormones have systemic effects beneficial for the treatment of diabetes mellitus (Type 1 and Type 2) (Unger, J., Curr Diab Rep., 2013; 13(5):663-668). Benefits may include augmentation of many aspects of beta ((β) cell function and number, suppression of glucagon secretion, increases in the insulin sensitivity of peripheral metabolic tissues, reduction of hepatic gluconeogenesis, and reduction of appetite. Two classes of incretin-based therapeutic agents have been identified for the treatment of diabetes mellitus (GLP-1 receptor agonists and dipeptidyl peptidase 4 (DPP-4) inhibitors). However, there is currently no incretin-based cell therapy option that would encompass an endogenous and cellular barometer for improved and efficient GLP1-based diabetes treatment. Furthermore, current incretin-based therapies are not regulated by circulating blood glucose levels and thus provide non-physiologically regulated GLP production.
In vertebrate embryonic development, a pluripotent cell gives rise to a group of cells comprising three germ layers (ectoderm, mesoderm, and endoderm) in a process known as gastrulation. The mesenchyme tissue is derived from the mesoderm, and is marked by the genes heart and neural crest derivatives expressed 1 (HAND1), and forkhead box F1 (FOXF1), among others. Tissues such as, thyroid, thymus, pancreas, gut and liver, will develop from the endoderm, via an intermediate stage. The intermediate stage in this process is the formation of the definitive endoderm. By the end of gastrulation, the endoderm is partitioned into anterior-posterior domains that can be recognized by the expression of a panel of factors that uniquely mark anterior foregut, posterior foregut, midgut, and hindgut regions of the endoderm.
The level of expression of specific transcription factors (“TFs”) may be used to designate the identity of a tissue, as described in Grapin-Botten et al., Trends Genet, 2000; 16(3): 124-130. FOXA2 marks the entire endoderm along the anterior-posterior axis. During transformation of the definitive endoderm into a primitive gut tube, the gut tube becomes regionalized into broad domains that can be observed at the molecular level by restricted gene expression patterns. The anterior foregut is marked broadly by the high expression of SOX2, and encompasses organ domains such as the thyroid, lung, and esophagus. The midgut (includes the duodenum, ileum, jejunum) and hindgut (includes the colon) are marked by high expression of caudal type homeobox 2 (CDX2). The SOX2-CDX2 boundary occurs within the posterior foregut, within which additional TFs mark specific organ domains. The regionalized pancreas domain within the posterior foregut shows a very high expression of PDX1 and very low expression of CDX2 and SOX2. PTF1A is highly expressed in pancreatic tissue. Low PDX1 expression, together with high CDX2 expression marks the duodenum domain. The intestinal endoderm is patterned by specific homeobox (HOX) genes. For example, HOXC5 is preferentially expressed in midgut endoderm cells. In addition, the expression of HOXA13 and HOXD13 are restricted to hindgut endoderm cells. The ALB gene, or albumin 1 protein, marks the earliest liver progenitors in the posterior foregut endoderm (Zaret et al., Curr Top Dev Biol, 2016; 117:647-669).
Strides have been made in improving protocols to generate intestinal endoderm cells from human pluripotent stem cells. For example, the following publications (Spence et al., Nature, 2011; 470(7332):105-109; Watson et al., Nature Medicine, 2014; 20(11):1310-1314; and Kauffman et al., Front Pharmacol, 2013; 4(79):1-18) outline differentiation protocols using either fibroblast growth factor (FGF)-4, Wingless-type MMTV integration site family, member 3A (WNT3A), Chiron 99021, or retinoic acid (RA) and FGF7 starting at the definitive endoderm stage, that generate mid-/hindgut spheroids, containing not only a CDX2+/FOXA2+ endodermal population, but also a significant mesenchymal CDX2+ cell population. The process of differentiating enteroendocrine cells from these hESC-derived mid-/hindgut spheroids is very inefficient, requiring a long time period, and is directed non-discriminately towards the generation of all intestinal cell types of the intervillus and villus regions. A need still exists for technology to generate intestinal midgut endoderm cells, without substantial contaminating mesenchyme, so as to be able to produce with high efficiency intestinal enteroendocrine cells for cell therapeutics.